Sexual transmission of human immunodeficiency virus type-I (HIV-1) accounts for the vast majority of new infections worldwide. In most cases, infection occurs as a result of interaction of the virus with CD4+ target cells in the mucosa or sub-epithelium. Interaction of HIV- 1 with CD4 on the cell surface is insufficient for viral entry; however, indicating that additional factors are required for infection to take place. Recent studies have identified several related seven- transmembrane receptors that function as co-factors for viral fusion and entry. Although these co-receptors are utilized by laboratory adapted strains of HIV-1, the extent to which they are used by primary isolates of HIV-1 remains largely unknown. It is of particular importance for vaccine development to determine whether these receptors are used by sexually transmitted strains of HIV-1, whether certain co- receptors are used preferentially and whether this process can be inhibited by the natural ligands for these receptors. The studies outlined in this proposal will directly evaluate the co-receptor requirements of transmitted strains of HIV-1 using a well-characterized panel of primary viruses isolated from patients during acute HIV-1 infection. In preliminary studies, we have evaluated co-receptor use by primary isolates of HIV-1 using a panel of CD4+ cell lines stably expressing each of the known HIV-1 co-receptors. In addition, we have developed a single-cycle complementation assay to determine whether interaction of the viral envelope glycoproteins with each of the HIV-1 co-receptors is sufficient to mediate entry into CD4+ cells. Using these assays, the goals of the proposal are: 1) To determine the primary co-receptor(s) used by transmitted strains of HIV-1 and to correlate co-receptor use with viral phenotype and sensitivity to beta-chemokine blocking. 2) To determine whether primary, transmitted strains of HIV-1 mediate entry into CD4+ cells by interaction of their envelope glycoproteins with specific co-receptors. 3) To determine whether selection for a particular viral phenotype during transmission correlates with co-receptor expression on CD4+ target cells.